This invention was made with U.S. Government support under Contract No. DE-AC06-76RLO 1830 awarded by the U.S. Dept. of Energy to Battelle Memorial Institute. The U.S. Government has certain rights in the invention.
This invention relates to mass spectrometry and more particularly to a method for detection and analysis of multiple-charged ions and dissociated fragments thereof.
The analytical ability of mass spectrometry for large molecules has been greatly extended by techniques such as electrospray ionization which can produce intact molecular ions of high charge states (see R. D. Smith et al. "New Developments in Biochemical Mass Spectrometry: Electrospray Ionization" Analytical Chemistry, Vol. 62 (1990), pp. 882-889). In a normal ESI mass spectrum of a large molecule a distribution of charge states are formed. Since only m/z is measured, the molecular weight is calculated using the multiple m/z measurements which are known to differ by a charge of 1 due to the quantum nature of electronic charge. The calculation is straightforward since there are only two unknowns (m and z) and an abundance of m/z measurements.
In tandem mass spectrometry, however, the dissociation of only a specific charge state of the molecular ion is examined. Thus, while m and z of the "parent" ion are known (from the initial "conventional" ESI-mass spectrum), interpretation of the "daughter" ions formed from dissociation of a single parent charge state generally do not provide any such features. Thus, interpretation of daughter ion spectra in tandem MS/MS studies is problematic.
Two major problems remain to be solved to effectively exploit these techniques in important chemical and biological applications.
Improvements are needed in sensitivity so that femtomole (10.sup.-15 mole) and, ideally, attomole (10.sup.-18 mole) quantities of a molecular species can be analyzed by the methods of tandem mass spectrometry. In tandem mass spectrometry, intact molecular ions selected from a primary mass spectrum are caused to dissociate, due to either collisional or photo-induced activation, to yield structurally-informative fragment, or daughter, ions, which are analyzed in a second analyzer. Developments in simultaneous ion detection, using an array detector, have improved detection sensitivity over scanning mode detection (see G. J. Louter et al., "A Very Sensitive Electro-Optical Simultaneous Ion Detection System" Internat. J. of Mass Spectrometry and Ion Processes, Vol. 50 (1983), pp. 245-257 and C. E. D. Ouwerkerk et al. "Simultaneous Ion Detection in a Double Focusing Mass Spectrometer with Specially Shaped Magnetic Pole Faces" Internat. J. of Mass Spectrometry and Ion Processes, Vol. 70 (1986) pp. 79-96). Nonetheless, studies with singly charged ions, which until very recently were the only case being studied, are limited to maximum molecular weights of about 3000.
An improved method is needed for accurate assignment of charge and mass assignment to the daughters produced by the dissociation of multiple-charged parent ions. Mass spectrometers separate according to mass-to-charge ratios (m/z), not mass. For single-charged ions, interpretation is trivial. Recent development of an analytic technique called charge-separation mass spectrometry has extended the interpretation to the dissociation of double-charged ions (see J. H. D. Eland, "A New Two-Parameter Mass Spectrometry" Acc. Chem. Res., Vol 22, No. 11, 1989, pp. 381-387) and of triply-charged ions (see D. A. Hagan and J. H. D. Eland, "Charge Separation of Triply Charged Ions" Rapid Communications in Mass Spectrometry, Vol. 3, No. 6, 1989, pp. 186-189). This technique employs single-stage time-of-flight mass spectrometry to obtain two-dimensional multi-ion coincidence spectra. So far, however, this technique is limited in application to dissociations inherent in the ionization process. The reported studies have been limited to simple molecular ion (CS.sub.2 and C.sub.6 D.sub.6) which present no ambiguities in assigning charge states to fragment ions. Double, triple and occasional quadruple-charged parent ions of relatively small molecules principally dissociate into neutral, single and double-charged fragment ions via a limited number of fragmentation pathways. These present little or no ambiguity in charge assignment. Stable or metastable triple-charge ions observed are a very small proportion of triply charged ions originally formed and are essentially ignored.
For dissociation of large, multiple-charged parent ions, there are virtually innumerable potential fragmentation pathways. Such dissociations yield more highly-charged daughter ion products, the charges of which are unknown, and many possible mass-to-charge ratios. The combinations of all these possibilities lead to severe ambiguities in charge and mass assignment. This situation has prevented application of prior art techniques to most analytical problems of real interest.
Accordingly, a need remains for an effective way to analyze complex molecules and ions.